Molecular basis for ionic strength dependence and crystal morphology in two-dimensional streptavidin crystallization

The two-dimensional crystallization of streptavidin at functionalized lipid interfaces is one of the best studied model systems for investigating molecular self-assembly processes. This system also provides an opportunity to elucidate the relationship between protein-protein molecular recognition, crystallization solution conditions, and crystal properties such as coherence, space group symmetry, and morphology. A better understanding of these relationships may aid in the design of rational strategies for promoting high-quality protein crystallization and for controlling protein assembly at interfaces in the biomaterials and nanotechnology fields. Here we show that two-dimensional streptavidin crystallization is kinetically controlled and that formation of a single electrostatic interaction at the crystal contact interfaces is a key energetic determinant of the kinetic barriers controlling crystal morphology. Our results also demonstrate that this electrostatic interaction at the streptavidin protein-protein interfaces is responsible for the ionic strength dependence of streptavidin crystallization. Molecular modeling studies of the wild-type crystal that displays C222 symmetry suggested that the side-chain amines of lysine 132 from adjacent proteins interact with each other across the dyad-related crystal contacts. Leucine was substituted at this position (K132L) to remove the need for bridging counterions. Unlike wild-type streptavidin, the K132L mutant crystallizes with rectangular morphology on buffer or on a pure water subphase and analysis of the electron micrographs demonstrates that the crystal retains C222 symmetry in the presence or absence of salt. The kinetic barriers associated with formation of this electrostatic interaction thus underlie the wild-type butterfly crystal morphology.